Fig 4. VEGF inhibitors rescue overangiogenesis and leakage in the CoCl₂-induced hypoxic retinal vasculature.

(A) Three conditions were designed to mimic the clinical situation and to examine the effects of treatment. Method I: Tg(fli1a:EGFP) embryos were treated with CoCl₂ (black line) and the inhibitor (red dotted line) simultaneously from 1 dpf to 5 dpf. Method II: Prior to CoCl₂ treatment (black line) at 3 dpf, we immersed embryos in the inhibitor (red dotted line) for 2 days. Method III: Following CoCl₂treatment, the inhibitor was added to the solution at 3 dpf. All embryos were injected with TAMRA dye at 4 dpf, and their retinal vasculatures were subsequently observed at 5 dpf. (B) CoCl₂-treated embryos showed TAMRA dye seepage (red) from the intraocular vessels (green). Similar leakages and excessive neovascularization were appeared in the control IgG-treated groups (CoCl₂ and normal human control IgG cotreatment). Three candidate inhibitors, SU5416, bevacizumab, and ranibizumab, reversed the effect of CoCl₂ on retinal vessels. (C) Statistical analyses of branch points showed significant rescue in inhibitor-treated embryos. The total numbers of embryos for analyses are indicated on the top of each bar. (D) However, the survival rates (four independent experiments, n = 35 in each) showed low toxicity under the Method I condition. Each bar represents the mean ± SEM of * (p < 0.0125) compared with the CoCl₂ = 5 mM group.